Human cancers vary markedly from patient to patient with respect to growth, aggressiveness and response to radiation therapy, chemotherapy and surgery. This might be due to the effect that microenvironmental factors such as hypoxia has on the expression of oncogenes, cytokines and angiogenic factors. This has led to a number of oxygen regulated protein expression in tumor cell lines in vitro and in vivo. However, the results do not adequately account for the expression of oxygen regulated proteins (ORPs) such as vascular endothelial growth factor and metallothionein (MT) in human squamous cell carcinomas in clinical studies. Evidence is presented here that hypoxic cells in squamous cell carcinomas (SCCs) exhibit signs of terminal differentiation and it is hypothesized that genetic controls associated with terminal differentiation exert collateral control on the expression of oxygen regulated genes. The hypothesis will be tested in both specific and general ways. A specific test will use in situ hybridization analysis of MT mRNA isoform expression which changes from inducible MT-I mRNA to non-inducible MT-IV mRNA during epithelial cell differentiation. It is predicted that hMT-IV mRNA will be expressed in hypoxic cells of SCCs thereby accounting for the general lack of MTI(II) protein expression in these cells. A general test of the hypothesis will use powerful new DNA array technology. Gene expression for oxygen regulated proteins and energy metabolism, among others, will be compared in hypoxic and oxic regions of SCCs that have been microdissected from tissue sections. It is predicted that hypoxic cells in regions of terminal differentiation in SCCs will not express the ORP genes that are observed in hypoxic tumor cell lines in vitro. Pimonidazole binding will be used to identify regions of hypoxia; proliferating cell nuclear antigen (PCNA) will be used to locate regions of proliferation; and, involucrin will be used to identify regions of terminal differentiation in SCC tissue sections. While of interest to all cancer therapies, it is believed that the results of this study will be of particular relevance to conventional radiation therapy and radiation therapies that include adjuvants designed to deal with tumor hypoxia.